Effect of staurosporine on fMet-Leu-Phe-stimulated human neutrophils: dissociated release of inositol 1,4,5-triphosphate, diacylglycerol and intracellular calcium

Abstract

Staurosporine, a microbial alkaloid, enhances inositol 1,4,5-triphosphate (IP 3) and 1,2-diacylglycerol (DG) production rapidly and dose-dependently in fMet-Leu-Phe (FMLP)-stimulated human neutrophils showing maximal effects at 1 μM concentration. The IP 3 increase was specific for staurosporine as three other putative protein kinase C (PKC) inhibitors, H7, sphingosine and palmitoylcarnitine were unable to enhance the IP 3 generation in FMLP-stimulated human neutrophils. Staurosporine, at concentrations 0.3–1.0 μM, did not affect the initial mobilization of FMLP-induced intracellular Ca 2+ (Ca 2+ i), although a sustained elevation of cytosolic Ca 2+ level was observed within 5 min. This effect could not be suppressed, even by 1 μM phorbolmyristate 12,13-acetate (PMA). Whereas lower concentrations of staurosporine (≤ 100 nM) were unable to affect FMLP-induced IP 3 production, DG accumulation and Ca 2+ i, the PMA-inhibited initial Ca 2+ i signal and IP 3 formation triggered by FMLP were almost completely restored. At higher concentrations (≥ 300 nM) staurosporine reversed the inhibitory effect of other protein kinases, distinct from the PMA-inducible one, which may be responsible for the phosphatidyl inositol 4,5-biphospinate (PIP i) breakdown, thus causing accumulation of IP 3 and DG and an elevation of Ca 2+ i level. Whereas IP 3 declined to basal level within 5 min, the DG level remained elevated during the same period. This phenomenon is attributed to phospholipase D (PLD) stimulation by staurosporine, which augments the DG synthesis, in part through PA degradation via phosphatidic acid (PA) phosphohydrolase.